Fractionation and quantitative analysis of charged particles such as macromolecules has traditionally been a slow and expensive process. Tests utilizing immunochemistry have been successfully applied to single component analysis of substances having requisite properties, but these methods have not been applicable to separation and quantitation of multiple proteins species from a single sample. Such determinations continue to be carried out by electrophoresis (i.e., differential migration of charged molecules in an electrical field) which continues to be regarded as a high resolution research method not applicable to high volume testing.
Measurement of the level of cholesterol in human serum is one of the most commonly ordered diagnostic procedures. It is believed to be highly predictive of the probability of occurrence of coronary heart disease (CHD). Despite the fact that the National Cholesterol Education Program has stated that determination of the overall lipid profile (i.e., the specific distribution of the total cholesterol among its HDL, LDL, VLDL, and chylomicron forms) is more predictive of CHD than total cholesterol measurement, the number of lipid profile determinations carried out remains relatively small compared to the number of total cholesterol tests. This is largely due to the relative difficulty and expense of carrying out accurate lipid profile tests. Lipid profile tests have been conducted by ultracentrifuge, gel chromatography, fractional precipitation, and electrophoretic methods, but these have not been found to be cost effective and convenient for high volume testing outside of the laboratory.
Electrophoresis is applicable to these problems and continues to be a popular and powerful method for analyzing charged macromolecules despite its complexity. Variants of electrophoresis have been developed over the years which sought to incorporate the physical advantages of certain states and configurations of matter. Among these, gel, thin film, and capillary electrophoresis have made notable contributions in biochemical analysis.
Use of gels as electrophoretic media minimizes the convective and diffusive randomization of bands promoting more rapid separations. Thin film and capillary techniques have made use of thin layers allowing greater heat transfer, hence more rapid separation through the use of higher voltages. Despite their advantages in a research environment, these techniques have remained esoteric and difficult to implement outside a laboratory setting.
Current clinical methods for separating HDL (high density lipoprotein), LDL (low density lipoprotein), and VLDL (very low density lipoprotein) cholesterol fractions include ultracentrifugation, gel chromatography, fractional precipitation, and electrophoresis.
Ultracentrifugation is considered a reference method since the designations HDL, LDL, and VLDL actually refer to density fractions separated by centrifuge; however, overnight processing is required for each centrifugation step. Gel chromatography correlates well with centrifugal analysis and is more rapid, but requires equally exotic equipment and expertise.
Fractional precipitation is the most commonly used method for lipid profiling in the conventional clinical laboratory. This procedure is reasonably convenient in a laboratory setting, but has shortcomings in validity and accuracy. In the fractional precipitation procedure, LDL cholesterol is calculated from the equation: ##EQU1##
A direct analysis is performed for total cholesterol, HDL cholesterol, and triglycerides. HDL cholesterol is analyzed by fractional precipitation followed by enzymatic assay of the supernatant solution, while total cholesterol and triglycerides are analyzed directly by conventional enzymatic methods. VLDL cholesterol is assumed to be equal to 1/5 (triglycerides) and chylomicron cholesterol is assumed to be zero in fasting patients.
Thus, the fractional precipitation method commonly used has several disadvantages. First, it is based on assumptions that are only approximately correct under fasting conditions. Second, it requires a fasting patient. Third, it requires three separate analytical procedures which require independent calibration. Fourth, it is not easy to perform outside a laboratory environment.
Muniz (of Ames Co., a division of Miles Laboratories) has described a method for separating cholesterol fractions by conventional electrophoresis. Clin. Chem. 23/10, pp. 1826-1833 (1977), where a homogeneous running gel of polyacrylamide is used to run samples of plasma. The chylomicron fraction remained in the sample gel, the VLDL were retained near the origin of separation gel, and the LDL and HDL fractions were bands in the separation (or running) gel. Quantitation of the separate fractions is not readily obtained from such a system because the gels are fragile and cannot be removed.
Cobb and Sanders (of Helena Laboratories) described a method for quantitating separated cholesterol fractions from a thin layer electrophoresis plate, Clin. Chem. 24/7, pp. 1116-1120 (1978). The authors report having used cellulose acetate plates on a plastic backing; but this procedure is laborious, imprecise, and does not allow quantitation of VLDL cholesterol in many cases.
Boschetti (U.S. Pat. No. 4,189,370, issued Feb. 19, 1980) described a method for separation of cholesterol fractions using two different, contiguous electrophoretic zones in a thin layer configuration. This procedure separated only two of the four cholesterol fractions, and did not provide for quantitation of separated fractions.
Allen (U.S. Pat. No. 3,620,947, issued Nov. 16, 1971) described an electrophoretic device with a plurality of contiguous zones. This device did not demonstrate high resolving power for proteins, and no means was provided to allow removal of separated fractions for quantitation.
Stathkos (U.S. Pat. No. 3,844,925, issued Oct. 29, 1974) described an isoelectric focusing column with a plurality of particle bed zones interposed between liquid regions with ports for sampling. The particle beds were of identical composition and served as locations for developing the pH gradients required for isoelectric focusing. This device provided a sampling scheme for proteins having different isoelectric points but did not have size separation properties.
To date only ultracentrifuge and gel chromatography methods have afforded a complete separation of all cholesterol fractions, but a simple, convenient device or system for a total and direct lipid profile separation and analysis from a single sample has not been achieved.